Photoremovable caging groups find extensive use in many fields ranging from cell biology to materials science. The general requirement of UV or blue light is a significant limitation due to associated toxicity and poor tissue penetration. By contrast, light between 650 and 900 nm, often referred to as the near-IR window, is cytocompatible and has significant tissue penetration (centimeters). Useful single photon reactions in the near-IR range would allow photorelease or uncaging approaches to be applied in complex biological settings. My lab is approaching this task by using chemical reactions involved in the photodecomposition or photobleaching of near-IR fluorophores. These oft-encountered light-initiated processes can occur with rapid kinetics, often undesirably so, and I believe represent an untapped opportunity for chemical biology. Our current efforts in this area are split in three aims. Aim 1: Development and mechanistic studies of near-IR photorelease chemistry. Over the past year, we have developed an uncaging reaction sequence initiated by 690 nm light using readily synthesized C4'-dialkylamine-substituted heptamethine cyanines. We have shown that a variety of phenol- and amine- containing small molecules are quickly uncaged upon irradiation with low energy light. Detailed mechanistic studies involving mass spectrometry, NMR, and absorbance techniques have shown that release occurs through regioselective C-C cleavage and then hydrolysis of the C4'-amine. While the photooxidative cleavage reaction had been previously described in cyanine photobleaching literature, these efforts are the first to use it for productive application. We are currently broadening the scope of the release process and examining aspects of the mechanism in detail using computational (collaboration with Dr. Joseph Ivanic) and experimental techniques. Aim 2: Application of uncaging reactions for targeted drug delivery. Light is used in a variety of cancer treatments ranging from established therapeutic techniques, such as PDT, to emerging areas such as fluorescence-guided surgery. Separately, the last decade has seen the clinical success of a number of antibody-drug conjugates. An enduring challenge is the development of precisely controlled linker strategies. To address this, we are applying our light-cleavable chemistry for targeted drug delivery (collaboration with Dr. Hisataka Kobayashi, Dr. Peter Choyke). This approach would merge the unique potency of small molecule drugs with the high spatial control afforded by light release and molecular targeting. The use of tissue penetrant, cytocompatible near-IR light is critical because existing uncaging chemistries using UV or blue light would not be suitable for this application. Towards this goal, we have shown that cell viability can be inhibited through light-dependent release of several cytotoxic small molecules. Aim 3: Near-IR light control of gene expression. Among many strategies to achieve precise regulation of gene expression, several studies over the past 15 years have used UV light-mediated uncaging of small molecules in combination with inducible gene expression systems. It is quite likely that near-IR uncaging will prove beneficial as these techniques progress in complex biological settings and organismal contexts. We have shown that our approach can be used to regulate gene expression through uncaging of an estrogen-receptor antagonist in a ligand-dependent CreERT/LoxP-reporter cell line (collaboration with Dr. Susan Mackem). These results set the stage for further applications that combine near-IR uncaging with widely available CreERT-mediated recombination approaches.